Methods of treating a surface of a polymer material by atmospheric pressure plasma

ABSTRACT

A method for treating a flexible plastic substrate is provided herein. The method includes establishing an atmospheric pressure plasma beam from an inert gas using a power of greater than about 90W, directing the plasma beam toward a surface of the flexible polymer substrate, and scanning the plasma beam across the surface of the polymer substrate to form a treated substrate surface.

RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 62/578,020 filed on Oct. 27, 2017 the contents ofwhich are relied upon and incorporated herein by reference in theirentity as if fully set forth below.

FIELD

The present disclosure generally relates to treating a surface of apolymer material. In particular, the present disclosure relates toatmospheric pressure plasma treatment for surface modification of apolymer substrate.

BACKGROUND

Bags containing fluids under sterile conditions are used in thebioprocess industry for the formulation, storage, transfer and transportof fluid while maintaining sterile conditions. Similarly, bags used tohold blood, urine and other materials utilized for medical purposespreferably provide and maintain sterile conditions for said materials.The bags may be used for biological fluids such as serum, buffers, andultrapure water and also for growing cell cultures to obtain thevaluable biopharmaceutical compounds produced by cells. Some of thecharacteristics of the bags to preserve the quality of the productscontained within include biocompatability with the products, sterility,and non-pyrogenicity. The bags are typically disposed of after use andare recognized as efficient means to prepare and store sterile fluids.Generally, these disposable bags are flexible and made from compatibleplastic that is sterilized by Gamma radiation.

Such bags typically include a single material or a laminate of materialsfolded or cut and sealed to provide a container or vessel to hold liquidmedium and cells. The bags are generally flexible and disposable. But,while conventional flexible and disposable bags may be inexpensive andadaptable, such bags may contaminate, to some degree, biologicalmaterial constituents processed therein via extractables and leachablesfrom the polymers, particularly if the biological materials are causticor if processing within the bags, or manufacture of the bags, is done atelevated temperatures, which may impact processing results if thebiological materials are sensitive to polymeric extractables andleachables.

SUMMARY

According to embodiments of the present disclosure, a method fortreating a flexible plastic substrate is provided. The method includesestablishing an atmospheric pressure plasma beam from an inert gas usinga power of greater than about 90W, directing the plasma beam toward asurface of the flexible polymer substrate, and scanning the plasma beamover at least a portion of the surface of the polymer substrate to forma treated substrate surface.

Additional features and advantages will be set forth in the detaileddescription which follows, and in part will be readily apparent to thoseskilled in the art from that description or recognized by practicing theembodiments as described herein, including the detailed descriptionwhich follows, the claims, as well as the appended drawings.

It is to be understood that both the foregoing general description andthe following detailed description are merely exemplary, and areintended to provide an overview or framework to understanding the natureand character of the claims. The accompanying drawings are included toprovide a further understanding, and are incorporated in and constitutea part of this specification. The drawings illustrate one or moreembodiment(s), and together with the description serve to explainprinciples and operation of the various embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will be understood more clearly from the followingdescription and from the accompanying figures, given purely by way ofnon-limiting example, in which:

FIG. 1 is a flow chart illustrating a method for treating a surface of apolymer material in accordance with embodiments of the presentdisclosure;

FIG. 2 illustrates an exemplary atmospheric pressure plasma apparatus inaccordance with embodiments of the present disclosure;

FIG. 3 illustrates an exemplary atmospheric pressure plasma apparatus inaccordance with embodiments of the present disclosure;

FIG. 4 illustrates an exploded view of an exemplary bioprocess bag inaccordance with embodiments of the present disclosure;

FIG. 5 illustrates an exploded view of an exemplary bioprocess bag inaccordance with embodiments of the present disclosure; and

FIG. 6 shows timed wavelength chromatograms of Ultra Performance LiquidChromatography (UPLC) for: a background reading; a glass sample; aMetallocene film treated with 20 scans of helium plasma at a power of120W; a Metallocene film treated with 40 scans of helium plasma at apower of 150W; a Metallocene film treated with 80 scans of helium plasmaat a power of 150W; four separate 10L Metallocene plastic bags; and aBis(2,4-ditert-Butylphenyl) phosphate (bDtBPP) standard.

DETAILED DESCRIPTION

Reference will now be made in detail to the present embodiment(s), anexample(s) of which is/are illustrated in the accompanying drawings.Whenever possible, the same reference numerals will be used throughoutthe drawings to refer to the same or like parts.

The singular forms “a,” “an” and “the” include plural referents unlessthe context clearly dictates otherwise. The endpoints of all rangesreciting the same characteristic are independently combinable andinclusive of the recited endpoint. All references are incorporatedherein by reference.

As used herein, “have,” “having,” “include,” “including,” “comprise,”“comprising” or the like are used in their open-ended sense, andgenerally mean “including, but not limited to.”

All scientific and technical terms used herein have meanings commonlyused in the art unless otherwise specified. The definitions providedherein are to facilitate understanding of certain terms used frequentlyherein and are not meant to limit the scope of the present disclosure.

The present disclosure is described below, at first generally, then indetail on the basis of several exemplary embodiments. The features shownin combination with one another in the individual exemplary embodimentsdo not all have to be realized. In particular, individual features mayalso be omitted or combined in some other way with other features shownof the same exemplary embodiment or else of other exemplary embodiments.

Embodiments of the present disclosure relate to methods for treating thesurface of a polymer substrate and to bioprocess bags including polymermaterials having at least a portion of a surface treated in accordancewith the present disclosure. Methods described herein include treating apolymer substrate surface with atmospheric pressure plasma to form atreated surface. As compared to an untreated polymer substrate surfacethe treated polymer substrate surface has a reduced overallconcentration of extractable and leachable compounds. In particular, ascompared to an untreated polymer substrate surface the treated polymersubstrate surface has a reduced concentration ofBis(2,4-ditert-Butylphenyl) phosphate (bDtBPP). For example, treatedpolymer substrate surfaces formed in accordance with embodiments of thepresent disclosure exhibit a reduction of greater than about 70% totalextractable and leachable content as compared to an untreated polymersubstrate surface.

FIG. 1 depicts a flow chart for a method 100 for treating a surface of apolymer substrate in accordance with embodiments of the presentdisclosure. As described herein, the polymer substrate may be a film orlaminate that includes at least one plastic material from the followinggroup: polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC),polyethylene terephtalate (PET), polystyrene (PS), polycarbonate (PC),polymethylpentene (PMP), polyetheretherketone (PEEK)polytetrafluoroethylene (PTFE), polyethylene-co-vinyl acetate (EVA),polyfluoroalkoxy (PFA) and derivatives thereof. The film or laminate mayhave a low melting temperature and may also have high gas permeability.

According to embodiments of the present disclosure, the method 100 mayinclude establishing 110 an atmospheric pressure plasma beam from aninert gas. As used herein, the term “atmospheric pressure plasma”includes a plasma formed from an inert gas under ambient pressureconditions, for example a pressure of about 1 atmosphere (atm), or atpressures ranging from about 0.1 atm to about 5 atm. Exemplary inertgasses include one or more of helium (He), neon (Ne), argon (Ar),krypton (Kr), xenon (Xe), and Radon (Rn), and optionally, hydrogen (H2),nitrogen (N2), or air and any combinations thereof at any desiredratios. The electrical power used to excite the plasma to form theatmospheric pressure plasma beam may be greater than about 90W, forexample between about 90W and about 250W, or between about 120W andabout 220W, or even between about 140W and about 200W, and all valuestherebetween.

According to embodiments of the present disclosure, the method 100 mayfurther include directing 120 the plasma beam toward a surface of theflexible polymer substrate to expose a portion of the surface of theflexible polymer substrate to the plasma beam.

According to embodiments of the present disclosure, the method 100 mayfurther include scanning 130 the plasma beam over at least a portion ofthe surface of the flexible polymer substrate to form a treatedsubstrate surface. Scanning 130 the plasma beam may include moving theplasma beam in relation to the flexible polymer substrate or moving theflexible polymer substrate in relation to the plasma beam. While theplasma beam may be moved over almost the entire surface area of thepolymer substrate surface, the method may alternatively include movingthe plasma beam over only a portion of the polymer substrate surface.For example, where the flexible polymer substrate is intended to be usedto form a single use flexible bag, such as a bioprocess bag, the methodmay include moving the plasma beam over only the portions of the polymersubstrate surface that will serve as the fluid contact layer of flexiblebag. The plasma beam may not be moved over other portions of the polymersubstrate surface, such as portions of the edges that may be welded toform the flexible bag. Optionally, scanning 130 the plasma beam mayinclude moving the plasma beam over a first portion of the polymersubstrate surface and then subsequently moving the plasma beam over asecond portion of the polymer substrate surface.

As described herein, a single scan describes the movement of the plasmabeam over a portion of the surface of the flexible polymer substrate.Subsequent movement of the plasma beam over the same portion of thesurface of the flexible polymer substrate is described as a subsequentscan of the polymer substrate surface. According to embodiments of thepresent disclosure, scanning 130 the plasma beam over at least a portionof the surface of the flexible polymer substrate to form a treatedsubstrate surface may include a plurality of scans over the same portionof the surface of the flexible polymer substrate. Scanning 130 theplasma beam over at least a portion of the surface of the flexiblepolymer substrate to form a treated substrate surface may includegreater than about 10 scans, or greater than about 20 scans, or greaterthan about 30 scans, or greater than about 40 scans, or greater thanabout 50 scans, or greater than about 60 scans, or greater than about 70scans, or greater than about 80 scans, or greater than about 90 scans,or even greater than about 100 scans. For example, scanning 130 theplasma beam over at least a portion of the surface of the flexiblepolymer substrate to form a treated substrate surface may includebetween about 10 scans and about 100 scans, or between about 20 scansand about 90 scans, or between about 30 scans and about 80 scans, oreven between about 40 scans and about 70 scans.

In addition to the number of scans over the same portion of the surfaceof the flexible polymer substrate, the portion of the surface of theflexible polymer substrate may be exposed to the plasma beam for anyperiod of time sufficient to form a treated substrate surface. Theportion of the surface of the flexible polymer substrate may be exposedto the plasma beam for greater than about 0.5 seconds, for example,between about 0.5 seconds and about 5.0 minutes, or between about 1.0second and about 3.0 minutes, or between about 3.0 seconds and about 1.0minute, or even between about 5.0 seconds and about 30 seconds, and allvalues therebetween. It is recognized that the volume of flexiblepolymer substrate intended to be treated may define or limit the numberof scans and the period of time the flexible polymer substrate may beexposed to the plasma beam. For example, where high volumes of flexiblepolymer substrate are intended to be treated, it may be advantageous toexpose the flexible polymer substrate to the plasma beam for arelatively shorter period of time while limiting the number of scansover the same portion of the surface of the flexible polymer substrate.

FIG. 2 shows an atmospheric pressure plasma apparatus 200 in accordancewith embodiments of the present disclosure. The apparatus 200 may be,for example, a dielectric barrier discharge (DBD) type plasma apparatusand generally includes a substrate support 202 and a plasma generator222. The apparatus 200 may include a substrate support 202. Thesubstrate support 202 may be any suitable support which positions theflexible polymer substrate such that the flexible polymer substrate canbe exposed to the atmospheric pressure plasma beam. As an example, FIG.2 includes a substrate support 202 that is rotatable about a centralaxis 204 such that a flexible polymer substrate present on the substratesupport 202 passes under a plasma outlet 206 of the plasma generator222. The apparatus 200 may include a distance between the substratesupport 202 and the plasma generator 222 of greater than about 0.5 mm.For example, the distance between the substrate support 202 and theplasma generator 222 may be between about 0.5 mm and about 10 mm, orbetween about 1.0 mm and about 5.0 mm, or between about 1.5 mm and about4.0 mm. Without wishing to be bound by any particular theory, it isbelieved that if the plasma generator 222 is placed a distance greaterthan about 10 mm from the substrate support 202 will prevent enoughdensification of the surface of the polymer film to form a treatedsubstrate surface as described herein.

FIGS. 2 and 3 show an atmospheric pressure plasma apparatus 200including one plasma generator 222, however, it should be appreciatedthat atmospheric pressure plasma apparatuses 200 according toembodiments of the present disclosure may include any number of plasmagenerators. More than one plasma generator 222 may allow for increasedexposure of the flexible polymer substrate to the atmospheric pressureplasma beam which may advantageously reduce the period of timesufficient to form a treated substrate surface. Alternative substratesupports 202 are also contemplated herein. For example, the apparatus200 may include a conveyor belt (not shown) where the flexible polymersubstrate moves on the conveyor belt past the plasma outlet 206 toexpose a surface of the flexible polymer substrate to the atmosphericpressure plasma.

Optionally, as shown in FIG. 3, an atmospheric pressure plasma apparatus300 in accordance with embodiments of the present disclosure may furtherinclude a first spool 302 and a second spool 304. In this optionalconfiguration, the first spool 302 includes the flexible polymersubstrate 322 wrapped on the spool. The substrate support 202 isrotationally driven in the direction indicated by arrow A in FIG. 3, thefirst spool 302 rotates in the direction indicated by Arrow B in FIG. 3,and the second spool 304 rotates in the direction indicated by Arrow Cin FIG. 3. Thus, the flexible polymer substrate 322 is wound from firstspool 302, around substrate support 202 and taken up on second spool 304as a treated flexible polymer substrate 324. Rollers may be disposedbetween the substrate support 202 and each of the spools 302, 304 tofacilitate movement of the flexible polymer substrate 322 or treatedflexible polymer substrate 324 off of, or on to, the spools 302, 304. Asshown in FIG. 3, the flexible polymer substrate 322 is wound around thesubstrate support 202 as the substrate support 202 rotates. The flexiblepolymer substrate 322 passes under a plasma outlet 206 of the plasmagenerator 222.

It is contemplated that other configurations of spools 302, 304 arepossible. For example, the spools may be disposed on any side of thesubstrate support 202 and the substrate support 202 may rotate in anydirection to facilitate plasma treatment of the flexible polymersubstrate 322. Additionally, while the spools 302, 304 are illustratedin FIG. 3 as being on the same side of the substrate support 202, thefirst spool 302 may be disposed on a first side of the substrate support202 and the second spool 304 may be disposed on a second side of thesubstrate support 202. Furthermore, with reference to FIG. 3, forpurposes of clarity, the flexible polymer substrate is described as aflexible polymer substrate 322 prior to being passed under the plasmaoutlet 206 of the plasma generator 222 and as a treated flexible polymersubstrate 324 after being passed under the plasma outlet 206 of theplasma generator 222. It should be appreciated that the flexible polymersubstrate may be passed under the plasma outlet 206 of the plasmagenerator 222 more than once. In doing so the substrate support 202 maybe rotationally driven in the direction opposite to the directionindicated by arrow A in FIG. 3, the first spool 302 may rotate in thedirection opposite to the direction indicated by Arrow B in FIG. 3, andthe second spool 304 may rotate in the direction opposite to thedirection indicated by Arrow C in FIG. 3. Thus, the flexible polymersubstrate 322 may further be wound from second spool 304, aroundsubstrate support 202 and taken up on first spool 302. The flexiblepolymer substrate 322 may be passed under the plasma outlet 206 of theplasma generator 222 any number of times.

The atmospheric pressure plasma apparatus 200, 300 may further includean inert gas source 210 fluidly connected to an electrode in the plasmagenerator 222 and configured to provide an inert gas to the electrodewhere plasma is formed from the gases. Gas exiting the gas source 210 isdirected to the electrode in the plasma generator 222 where anatmospheric pressure plasma is formed. The atmospheric pressure plasmathen passes from the electrode to the plasma outlet 206 where it exitsthe plasma generator 222. The electrode in the plasma generator 222 maybe coupled to a power source 226. The power source 226 may be furthercoupled to a ground electrode 228 disposed in the substrate support 202.A capacitively coupled plasma may be formed between the electrode in theplasma generator 222 and the ground electrode 228. The power source mayprovide radio frequency (RF) or microwave frequency (MF) power. Adielectric layer 232 may be disposed between a substrate 234 and theground electrode 228. Alternatively, (not shown) the dielectric layermay be a plurality of dielectric layers disposed between each theelectrode 222 in the plasma generator 222 and the substrate 234. Suchconfiguration is merely exemplary and it should be appreciated thatother suitable configurations of a dielectric barrier dischargeatmospheric pressure plasma apparatus may be utilized.

The atmospheric pressure plasma apparatus 200 may further include acontroller 236 coupled to various components of the apparatus 200 andconfigured to control the operation of such components. Althoughschematically shown coupled to the substrate support 202, the controllermay be operably connected to any of the components, and any number ofthe components, of the apparatus 200, in order to control the apparatus200 in accordance with embodiments of the present disclosure. Thecontroller 236 generally includes a central processing unit (CPU) 238, amemory 240, and support circuits 242 for the CPU 238. The controller 236may control the apparatus 200 directly, or via other computers orcontrollers (not shown) associated with particular components of theapparatus 200. The controller 236 may be one of any form ofgeneral-purpose computer processor that can be used in an industrialsetting for controlling various chambers and sub-processors.

The memory, or computer-readable medium 240 of the CPU 238 may be one ormore of readily available memory such as random access memory (RAM),read only memory (ROM), floppy disk, hard disk, flash, or any other formof digital storage, local or remote. The support circuits 242 arecoupled to the CPU 238 for supporting the processor in a conventionalmanner. These circuits include cache, power supplies, clock circuits,input/output circuitry and subsystems, and the like. Methods asdescribed herein may be stored in the memory 240 as a software routinethat may be executed or invoked to turn the controller into a specificpurpose controller to control the operation of the apparatus 200 inaccordance with embodiments described herein. The software routine mayalso be stored and/or executed by a second CPU (not shown) that isremotely located from the hardware being controlled by the CPU 238.

In operation, the substrate 200 may be placed on the substrate support202 and rotated about the central axis 204. The plasma outlet 206 mayprovide the first process gas and the atmospheric pressure plasma may beformed by providing power from the power source 226 to the electrode ofthe plasma generator 222 and the ground electrode 228. The substrate 200may be exposed to the atmospheric pressure plasma for any desired numberof rotations about the central axis and/or any desired period of time.

The method for treating a surface of a polymer substrate as describedherein may be performed using an atmospheric pressure plasma apparatus200 such as the one shown in FIG. 2. As such, the flexible polymersubstrate to be treated in accordance with the method described hereinmay be positioned on a substrate support 202. Additionally, scanning 130the plasma beam over at least a portion of the surface of the flexiblepolymer substrate to form a treated substrate surface may includerotating the substrate support 202 about a central axis 204 such thatthe surface of the flexible polymer moves relative to the plasma outlet206. Alternatively, the method for treating a surface of a polymersubstrate as described herein may be performed using an atmosphericpressure plasma apparatus 300 such as the one shown in FIG. 3. As such,the flexible polymer substrate to be treated in accordance with themethod described herein may be positioned on a first spool 302.Additionally, scanning 130 the plasma beam over at least a portion ofthe surface of the flexible polymer substrate to form a treatedsubstrate surface may include rotating the first spool 302 and windingthe polymer substrate from the first spool 302 and around the substratesupport 202 such that the surface of the flexible polymer moves relativeto the plasma outlet 206.

Additionally, in accordance with the method as described herein,establishing an atmospheric pressure plasma beam may include flowing aninert gas from an inert gas source 210 to an electrode of a plasmagenerator 222.

FIG. 4 illustrates an exploded view of an exemplary bioprocess bag inaccordance with embodiments of the present disclosure. As shown, thebioprocess bag 100 includes at least two sheets 102, 104 formed fromfilms or laminates. The sheets 102, 104 may be welded along the edges ofthe sheets to form a pillow-shaped bag having an interior compartmentfor receiving fluid. Alternatively, as shown in FIG. 5, the bioprocessbag 200 may be a three-dimensional bag having a bottom portion 202, atop portion 204 and a sidewall 280 which define an interior compartmentfor receiving fluid. At least one of the bottom portion 202, the topportion 204 and the sidewall 280 is formed from a film or laminate. Thesidewall 280 may be formed from a single continuous piece of material.Alternatively, the sidewall 280 may include two or more panels joinedalong edges thereof. The sidewall 280 and/or the bottom portion 202and/or the top portion 204 may be formed from a single piece of materialwhereby boundaries of the various portions of the bioprocess bag 200 aredefined by folds or creases. Alternatively, one or more of the sidewall280, the bottom portion 202 and the top portion 204 may be formedseparately and joined along edges thereof such as by welding theportions together. The bioprocess bag 100, 200 is hermetically sealedand preferably provides a closed system for use in all phases ofprocessing fluid and/or other components. As used herein, the term“closed system” refers to a system sealed to ensure sterility of thecontents of the system and to limit or prevent the introduction ofcontaminants from the surrounding atmosphere.

Each of the sheets 102, 104 of the bioprocess bag 100 may be formed fromone or more of the same or different materials. Each of the top portion204, the bottom portion 202 and the sidewall 280 of the bioprocess bag200 may be formed from one or more of the same or different materials.Where opaque or colored materials are used, at least a portion of thebag may be substantially clear to allow for viewing of fluid and/orother components in the interior compartment of the bag. For example, aportion of any of sheets 102, 104 or at least a portion of any of topportion 204, bottom portion 202 and the sidewall 280 may be clear toprovide a window through which the interior compartment of the bag canbe viewed.

As used herein, the term “fluid” is used to denote any substance capableof flowing, such as liquids, liquid suspensions, gases, gaseoussuspensions, or the like, without limitation. The term “fluid and/orother components” is used throughout the present disclosure to refer tofluid which may include cell culture media having nutrients for cellgrowth, cells, byproducts of the cell culture process, and any otherbiological materials or components that may conventionally be added orformed in a bioprocess system. Bioprocess bags and other vesselsdescribed herein may include one or more cells or reagents.Additionally, the bags may include cell culture media. Cell culturemedia may be for example, but is not limited to, sugars, salts, aminoacids, serum (e.g., fetal bovine serum), antibiotics, growth factors,differentiation factors, colorant, or other desired factors. Commonculture media that may be provided in the bag includes Dulbecco'sModified Eagle Medium (DMEM), Ham's F12 Nutrient Mixture, MinimumEssential Media (MEM), RPMI Medium, and the like. Any type of culturedcell may be included in the bag including, but not limited to,immortalized cells, primary culture cells, cancer cells, stem cells(e.g., embryonic or induced pluripotent), etc. The cells may bemammalian cells, avian cells, piscine cells, etc. The cells may be ofany tissue type including, but not limited to, kidney, fibroblast,breast, skin, brain, ovary, lung, bone, nerve, muscle, cardiac,colorectal, pancreas, immune (e.g., B cell), blood, etc. The cells maybe in any cultured form in the bag including disperse (e.g., freshlyseeded), confluent, 2-dimensional, 3-dimensional, spheroid, etc. In someembodiments, cells are present without media (e.g., freeze-dried, inpreservative, frozen, etc.).

The bioprocess bag 100, 200 may be formed from materials that areconventionally associated with disposable products for bioprocessapplications. Any or all of the sheets 102, 104 of the bioprocess bag100, or the bottom portion 202, top portion 204 and sidewall 280 of thebioprocess bag 200, may be formed from a film or laminate that includesat least one plastic material from the following group: polyethylene(PE), polypropylene (PP), polyvinyl chloride (PVC), polyethyleneterephtalate (PET), polystyrene (PS), polycarbonate (PC),polymethylpentene (PMP), polyetheretherketone (PEEK)polytetrafluoroethylene (PTFE), polyfluoroalkoxy (PFA) and derivativesthereof.

It should be understood that the dimensions of the bioprocess bag 100,200 including both relative and absolute dimensions can be varied. Forexample, the bags may be configured to hold a volume of fluid and/orother components of about 1.0 ml, or about 5.0 ml, or about 10 ml, orabout 25 ml, or about 50 ml, or about 100 ml, or about or about 250 ml,or about 500 ml, or about 1000 ml, or about 2000 ml, or about 5000 ml,or about 10,000 ml, or even about 20,000 ml, as well as all volumestherein between.

Ultra high pressure liquid chromatography (UPLC) is a form of columnchromatography used to separate, identify, and quantify compounds. UPLCallows for separation and analysis of small particles both quickly andeffectively. Generally, liquid chromatography is the process of passinga mixture of particles to be separated through a column. This allows theanalyte, which is separated from a mixture, to be measured from othermolecules. The columns are filled with a packing material, known as thestationary phase. In UPLC a pump pushes the mixture, known as the mobilephase, through the columns. As the mobile phase passes through thestationary phase, a detector shows the retention times of the differentmolecules. Retention time varies depending on the interactions betweenthe stationary phase, the molecules being analyzed, and the solventused.

An Acquity UPLC H-Class System used with Waters Acquity UPLC Photo diodearray detector using Cortecs C18 2.1×100 mm 2.7 μm Column (commerciallyavailable from Waters Corporation, Milford, Mass.) was utilized toanalyze various films as described below. FIG. 6 shows timed wavelengthchromatograms of UPLC for: a background reading 600; a glass sample 610;a Metallocene film 620 treated with 20 scans of helium plasma at a powerof 120W; a Metallocene film 630 treated with 40 scans of helium plasmaat a power of 150W; a Metallocene film 640 treated with 80 scans ofhelium plasma at a power of 150W; four separate 10L plastic bags 650,660, 670, 680; and a Bis(2,4-ditert-Butylphenyl) phosphate (bDtBPP)standard 690. bDtBPP is an extractable compound which has been shown tobe highly detrimental to cell growth. The compound is derived from thebreakdown of tris(2,4-di-tert-butylphenyl)phosphite (trade name Irgafos168®), a common antioxidant additive present in many formulations ofpolyethylene (one of the polymers commonly used as the material fluidcontact layer in bioprocess containers such as flexible single usebioprocess bags). Cell growth experiments using several mammalian celllines and growth media spiked with bDtBPP have shown harmful effects atconcentrations well below the parts-per-million range. Cellular responseto bDtBPP is rapid, and results in a significant decrease inmitochondrial membrane potential. The migration of bDtBPP frompolyethylene-based films is shown to be time- and temperature-dependent.Further, experiments suggest that exposure of oxidized Irgafos 168 toionizing radiation (such as gamma irradiation) is an important conditionfor the generation of significant amounts of leachable bDtBPP.

Each of Metallocene film 620, Metallocene film 630, and Metallocene film640 were treated with an atmospheric pressure plasma beam formed with anexcitation power of greater than 90W. The inert gas used to form theplasma was helium with a gas flow rate of about 14.5 standard liter perminute (SLM). A high frequency plasma generator (13.56 MHz) was utilizedin a continuous/non-filamentary mode. The plasma generator included aplasma outlet which was placed between about 2.0 mm and about 3.0 mmfrom the surface of a substrate support. The substrate support was aroller and each of the films was individually placed on the surface ofthe roller to be treated.

As can be seen from FIG. 6, each of the Metallocene films treated at150W exhibited a decrease in the concentration of bDtBPP as compared tothe four separate 10L plastic bag 650, 660, 670, 680. The Metallocenefilm 630 treated with 40 scans of helium plasma at a power of 150Wexhibited a decrease in the concentration of bDtBPP as compared to thefour separate 10L Metallocene plastic bag 650, 660, 670, 680. TheMetallocene film 640 treated with 80 scans of helium plasma at a powerof 150W exhibited an even greater decrease in the concentration ofbDtBPP as compared to the four separate 10L Metallocene plastic bags650, 660, 670, 680. The UPLC timed wavelength chromatogram forMetallocene film 640 resembled the UPLC timed wavelength chromatogramfor the glass sample 610.

Because of its detrimental effects on cell growth, bDtBPP is the citedherein as showing important differences in comparing the UPLC timedwavelength chromatograms shown in FIG. 6. However, it should also benoted that many of the other extractable and leachable compoundsexhibited by the four separate 10L Metallocene plastic bag 650, 660,670, 680 are also substantially reduced or eliminated in the films 620,630, 640 treated in accordance with the present disclosure. In fact, thefilms 620, 630, 640 treated in accordance with the present disclosureexhibited a reduction of greater than about 70% total extractable andleachable content as compared to the four 10L Metallocene plastic bags650, 660, 670, 680. Thus, while embodiments of the present disclosurereduce the concentration of bDtBPP in polymer films, embodiments of thepresent disclosure also reduce the amount of other extractable andleachable compounds which may be harmful to the growth of at least somecells.

Without wishing to be bound by any particular theory, it is believedthat helium plasma treatment in accordance with the present disclosurepromotes cross-linking and densification of the surface of the polymerfilm which is intended to serve as the fluid contact layer of bioprocessbags formed from the polymer film. The helium plasma treatment isbelieved to reduce the presence of extractable and leachable compoundson the surface of the polymer film by removing low molecular weightspecies from the surface of the polymer film and by inducingcrystallinity changes on the surface of the polymer film. Generally, dueto the ionization energy created by the plasma treatment, more reactivespecies are generated. Such reactive species may promote crosslinkingwhich drives out smaller molecules from the surface of the polymer andforms a relatively dense polymer surface. Such dense surface preventsthe diffusion of oxygen which may result in the breakdown of TBPP.

While the present disclosure includes a limited number of embodiments,those skilled in the art, having benefit of this disclosure, willappreciate that other embodiments can be devised which do not departfrom the scope of the present disclosure.

1. A method for treating a flexible polymer substrate, the methodcomprising: establishing an atmospheric pressure plasma beam from aninert gas using a power of greater than about 90W; directing the plasmabeam from a plasma outlet toward a surface of the flexible polymersubstrate; and scanning the plasma beam over at least a portion of thesurface of the polymer substrate to form a treated substrate surface. 2.The method of claim 1, wherein establishing the atmospheric pressureplasma beam comprises using a power of between about 120W and about220W. 3-4. (canceled)
 5. The method claim 1, wherein scanning the plasmabeam over at least a portion of the surface of the polymer substratecomprises moving the plasma beam over a first portion of the polymersubstrate surface and then subsequently moving the plasma beam over asecond portion of the polymer substrate surface.
 6. The method of claim1, wherein scanning the plasma beam over at least a portion of thesurface of the polymer substrate comprises moving the plasma beam inrelation to the polymer substrate.
 7. The method of claim 1, whereinscanning the plasma beam over at least a portion of the surface of thepolymer substrate comprises moving the polymer substrate in relation tothe plasma beam.
 8. The method of claim 1, wherein the inert gascomprises at least one of helium, neon, argon, krypton, xenon, andradon.
 9. The method of claim 1, wherein directing the plasma beamtoward a surface of the flexible polymer substrate comprises exposingthe surface of the flexible polymer substrate to the plasma beam forgreater than about 0.5 seconds, between about 0.5 seconds and about 5.0minutes, between about 1.0 second and about 3.0 minutes, between about3.0 seconds and about 1.0 minute, or between about 5.0 seconds and about30 seconds. 10-13. (canceled)
 14. The method of claim 1, wherein theflexible polymer substrate is positioned on a substrate support.
 15. Themethod of claim 14, wherein the substrate support is rotatable about acentral axis, and wherein scanning the plasma beam over at least aportion of the surface of the polymer substrate comprises rotating thesubstrate support about the central axis such that the surface of theflexible polymer moves relative to the plasma outlet.
 16. The method ofclaim 1, wherein the flexible polymer is positioned on a spool.
 17. Themethod of claim 16, wherein the spool is rotatable about a central axis,and wherein scanning the plasma beam over at least a portion of thesurface of the polymer substrate comprises rotating the first spool andwinding the polymer substrate from the first spool and around asubstrate support such that the surface of the flexible polymer movesrelative to the plasma outlet.
 18. The method of claim 1, whereinestablishing an atmospheric pressure plasma beam comprises flowing theinert gas from an inert gas source to an electrode of a plasmagenerator.
 19. The method of claim 1, wherein the treated polymersubstrate surface comprises a total extractable and leachable content ofless than about 70% of the total extractable and leachable content of anuntreated polymer substrate surface.
 20. The method of claim 1, whereinscanning the plasma beam over at least a portion of the surface of thepolymer substrate comprises greater than about 10 scans over the sameportion of the surface of the polymer substrate, greater than about 40scans over the same portion of the surface of the polymer substrate,greater than about 60 scans over the same portion of the surface of thepolymer substrate, or greater than about 80 scans over the same portionof the surface of the polymer substrate. 21-23. (canceled)
 24. Themethod of claim 1, wherein scanning the plasma beam over at least aportion of the surface of the polymer substrate comprises between about20 scans and about 90 scans over the same portion of the surface of thepolymer substrate.
 25. A flexible polymer substrate treated by themethod of claim
 1. 26. A bioprocess bag comprising flexible polymersubstrate of claim
 25. 27. A bioprocess system configured for culturingcells, the bioprocess system comprising the flexible polymer substrateof claim 25.